CN1194095C - Process for transforming macroseed plant and its application - Google Patents
Process for transforming macroseed plant and its application Download PDFInfo
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- CN1194095C CN1194095C CNB011044284A CN01104428A CN1194095C CN 1194095 C CN1194095 C CN 1194095C CN B011044284 A CNB011044284 A CN B011044284A CN 01104428 A CN01104428 A CN 01104428A CN 1194095 C CN1194095 C CN 1194095C
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Abstract
The present invention relates to a method for converting plants with large seeds and an application thereof. The procedures of the present invention mainly comprise: seedlings generated by aseptic seeds are used as materials, coleoptile or cotyledon and spires are peeled, stem tips are naked and are used as conversion receptors, and purpose genes are led in an agrobacterium tumefaciens medium method; the converted seedlings are transplanted to flower pots, vermiculite is covered, culture solution is irrigated, selective agents are sprayed after plants grow out three to four young leaves, and transgenic plants which show resistance are screened. The present invention can effectively overcome genotype restriction, plants which can not regenerate plants easily generate transgenic plants in batches in a tissue culture method. The present invention has an important value to the genetic improvement of cereal crops, legume with large seeds, cucurbitaceae crops, cotton, sunflowers, drupe class fruit trees and trees with large seeds which have important economic values.
Description
Technical field of the present invention:
Technical field under the present invention is agricultural biological technical field and plant genetic engineering field.
Research background of the present invention:
Plant genetic conversion (Genetic transformation) or title transform and are meant that an extraneous nucleotide fragment changes vegetable cell or tissue over to, make its stable reservation and expression in recipient cell or regeneration plant, and pass to the offspring by sexual or vegetative propagation.By transforming, can change the gene of control specific trait over to existing plant variety, break the dysgenesia of original kind, shorten breeding cycle greatly, create the novel improved seeds that traditional breeding method is difficult to obtain.
To successfully introduce a certain plant to foreign gene, improve transformation frequency, just must find the optimal conversion method that is fit to this kind of plant.The ideal methods for plant transformation should possess following characteristics: (1) DNA transfer efficiency height; (2) can be with the quite high frequency fertile plant of regenerating; (3) the genotype dependency is little; (4) program simple, efficient, economical, can repeat; (5) convenient screening transformant or plant; (6) time of needing of tissue culture is short or without tissue culture, to reduce the generation of somaclonal variation.
Adopt the transgenic technology (agriculture bacillus mediated genetic transformation, particle gun blast technique and protoplastis are the genetic transformation of acceptor) commonly used can be, but can then depend on acceptor systems from the required genotype large batch of transfer-gen plant of regenerating effectively with exogenous gene transfered plant cell.Therefore, foundation can not be subjected to the high-efficient transgenic acceptor systems of genotype restriction, is exactly the important content of plant genetic engineering breeding area research.
The history in existing more than 30 year of research to the plant genetic transformation system.1980 mid-nineties 90s, the success of cultivating regeneration plant along with multiple protoplastis is that the genetic transformation work of acceptor is developed rapidly with the protoplastis.At the beginning of later stage in the 1980's and generation nineteen ninety, obtained multiple transfer-gen plant respectively with electrization, PEG revulsion etc.Selecting protoplastis for use is that acceptor carries out genetic transformation, though have simple to operate, transformation efficiency is high, transfer-gen plant results from single cell clone and collaborative transformation efficiency advantages of higher, but because protoplast regenerated plant is subjected to the genotype restriction very big, being difficult to go up used genotype with production is material, thereby causes this method generally not adopted.
1985, and Horsch etc. (Horsch, R.B. etc., 1985, Science, 227:1229-1231) initiative is by the tobacco leaf disc conversion method (leaf disc transformation) of agrobacterium tumefaciens mediation.The meaning of this method is: (1) needn't utilize protoplastis, suspended culture cell or the callus acceptor as transgenosis, can directly transform with plant tissue; (2) set up and utilize natural T-DNA from the short-cut method of agrobatcerium cell to vegetable cell transfer destination gene.
After this, the Study on Transformation of different plant tissues is carried out in succession.Make explant with plant leaf, petiole, cotyledon, cotyledon petiole, hypocotyl, stem, scape, stolon, stem tuber, shoot apical meristem, bud root, piece root, zygotic embryo or somatic embryo even mature seed etc. respectively, all successfully obtain transforming tissue or/and plant.Because monocotyledons is not the natural host of Agrobacterium, agriculture bacillus mediated method for transformation once be mainly used in dicotyledons (Hawes, M.C. etc., 1987, Plant Cell Rept., 6:287-290).But be accompanied by the understanding to the T-DNA transfer mechanism, people have particularly done big quantity research on the cereal crop in monocotyledons in recent years, and this technology has developed into the major technique that cereal crop transforms.
1987, the investigator of Cornell University at first designed a kind of particle gun, and Sanford etc. (Wang, YC. etc., 1988, Plant Mol.Biol. 11:433-439) transforms onion epidermis cell with particle bombardment and succeeds.This technology be used to very soon cereal crop and fabaceous genetic transformation (Wang, YC. etc., Plant Mol.Biol., 11:433-439), the principal recipient material is an embryo callus.From the 1980's ends to nineteen ninety for mid-term, adopt this kind method on corn, paddy rice, wheat and Soybean and Other Crops, to obtain transfer-gen plant, obtain than quantum jump (Saito etc., Europe Patent Application 672752) with the genetic transformation of the dicotyledons of Agrobacterium-mediated Transformation thereby make cereal crop and be difficult to.
With respect to direct conversion method, all higher by transfer-gen plant quality and fertility that agrobacterium-mediated transformation obtains, and exogenous origin gene integrator is gone into the more accurate (Hansen etc. of acceptor gene group, 1997, Proc.Natl.Acad.Sci.U.S.A., 94:11726-11730), be generally single copy or low copy, and meet mendel's law substantially; The dna fragmentation that shifts has particular end, and gene rearrangement is less; Transferable big segmental DNA.Therefore, agriculture bacillus mediated genetic transformation is extremely paid attention to.
In Study on Transformation for important farm crop, scholars have adopted several different methods, attempt to set up the ideal acceptor systems, but up to the present, though obtained multiple transfer-gen plant, most agricultural kinds are not set up the higher transformation system of efficient yet.In other words, effectively do not set up yet at the method for transformation of main farm produce plant variety.
Research with the agroinfection Sunflower Receptacle just began as far back as the 1970's.Nineteen eighty-three, Murai etc. have obtained to change the Sunflower Receptacle callus of Kidney bean storage protein gene.Continue after, Matzke and Goldsbrough have obtained the Sunflower Receptacle transformant with similar approach in succession.1987, Everett etc. utilized the Agrobacterium-mediated Transformation Sunflower Receptacle, have obtained transfer-gen plant.1992, it was acceptor that Bidney etc. get stripped Sunflower Receptacle apical meristem, with the particle gun bombardment, used agroinfection more earlier, significantly improved transformation frequency, but shortcoming was the inconvenience of drawing materials, and technical difficulty is big.Because the most kinds of Sunflower Receptacle are difficult for from cultured tissue and cell regeneration plant, thus set up new transformation system very necessary (Takeuchi, Y. etc., 1992, Plant Mol.Biol., 18:835-839).
Cotton is subject to agroinfection, but tissue and cell cultures difficulty are big.1987, Firoozabady etc. and Umbech etc. were acceptor with cotton cotyledon and hypocotyl respectively, adopted agrobacterium-mediated transformation that NPT II gene is imported culturing cell and obtains transfer-gen plant.At the beginning of generation nineteen ninety, the pest-resistant cotton of trans Bt gene enters field experiment.1991, and McCabe etc. (McCabe, DE. etc., 1991, III International Congressof Plant Mol.Biol.Tuscen AZ) with particle gun bombardment cotton shoot apical meristem, obtains transfer-gen plant.Its concrete grammar is: after the seed disinfection, remove the plumular axis of aseptic seedling, expose meristematic tissue, with the particle gun bombardment, cultivate into strain then after the incubated overnight.
Soybean transgene is operated in 1988 and makes a breakthrough, and Agracetus company (McCabe, DE. etc., 1988, Bio/technol. 6:923-926) obtains transfer-gen plant respectively with Monsanto company.What Agracetus company adopted is the particle gun blast technique, and target tissue is the primary meristem and the axillary meristem of soybean embryo, and the advantage of this system is more easily to obtain regeneration plant, but it is lower to obtain the frequency of complete transformant.Monsanto company utilizes agrobacterium-mediated transformation to carry out transgenosis, and explant is the cotyledon segment of firm chitting piece, with NPT II, GUS and glyphosate tolerance gene transfered cell and regeneration plant.Because many soybean varieties are not subject to agroinfection (Owens, LD. etc., 1985, Plant Physiol.77:87-94; Owens, LD. etc., 1988, Plant Physiol.88:570-573; Delzer, BW. etc., 1990, Crop Sci.30:320-322; Mauro, AO. etc., 1995, Crop Sci.35:1152-1156), so the conversion system of Monsanto company is only applicable to the minority soybean varieties.
After this, (Christou, P. etc., 1990 such as Christou, Trends in Biotech., 8:145-151) with special-purpose particle gun soybean transformation stem apex, (Finer, JJ. etc. such as Finer, 1990, Plant Mol.Rep. 9:189-194) transforms embryonal suspension cell with the Biolistics_ particle gun, and usefulness Bioli stics_ particle guns such as Sato transform the immature embryo stem apex and long-term splitted soybean suspended cell culture is all succeeded.In addition, (Dhir such as Dhir, SK. etc., 1991, Plant Cell Rep.10:106-110.) also obtains complete transformed plant with electric shocking method soybean transformation protoplastis, (Chee etc., 1994, United States Patent (USP): the 5376543) U.S.'s patent of invention that " adopts Agrobacterium intermediary method to transform plant germination seed " 1992 and twice application in 1994 respectively such as Chee.In the specification sheets of this patent, only reported the work of adopting the wild-type Agrobacterium-mediated Transformation to sprout 24-48 hour soybean and phaseolus vulgaris seeds acquisition dross plant.This patent is abandoned after a while.2000, Finer etc. reported employing supersound process-agroinfection soybean transformation cotyledon can obtain the result of good result (Finer, KR. etc., 2000, Lett Appl Microbiol., 30:406-410).
Peanut genetic transformation difficulty is big.Up to 1991, Lacorte etc. (Lacorte etc., 1991, Plant CellRep.10:354-357) just utilized Agrobacterium-mediated Transformation peanut explant to obtain transgenic callus.1993, and Ozias-Akins etc. (Ozias-Akins etc., 1993, Plant Sci. is that acceptor has obtained transfer-gen plant with the embryo callus 93:185-194).1997, Cheng etc. (Cheng etc., 1997, Plant Physiol., 115:971-980) and Eapen etc. obtain the peanut transfer-gen plant respectively, but the plant regeneration difficulty is subjected to the genotype restriction big.In research report in recent years, still there are shortcomings such as the plant regeneration difficulty is big, regeneration plant fertility difference in used peanut trans genosis receptor system.
Be that the rice conversion of acceptor was operated in for 1980 mid-nineties 90s and has obtained transformant with the protoplastis, but up to 1988, Zhang etc. (Zhang, W. etc., 1988, Theor.Appl.Genet. 76:835-840) just adopts electric shocking method to obtain transfer-gen plant.After this, similar report is a lot, but the working efficiency of acquisition transfer-gen plant is not high.1992, and Cao etc. (Cao, J. etc., 1992, Plant Cell Rept. 11:586-591) adopts the particle gun blast technique to obtain the transfer-gen plant of antiweed.After this, be acceptor with the rataria, all succeed in a plurality of laboratories that are operated in of adopting the particle gun blast technique to obtain transformed plant, and this method also becomes the main method of nineteen ninety for rice genetic conversion in mid-term.
Utilize the research of Agrobacterium-mediated Transformation paddy rice to start from the later stage in the 1980's.Nineteen ninety, the wild-type Agrobacterium that usefulness Syringylethanone pre-treatment such as Raineri have wide host range infects the paddy rice scultellum then, detects T-DNA and integrate in rice cell in the inductive callus.1993, Chan etc. infected the rataria of japonica rice with Agrobacterium, obtain transfer-gen plant, but transformation frequency is lower.Next year, (Hiei, Y. etc., 1993, european patent application: 604662 such as Hiei; 1997, United States Patent (USP): 5591616; 1994, The Plant J. 6:271-277) has made up efficient conversion carrier, be used in combination Syringylethanone, set up the method that Agrobacterium efficiently transforms japonica rice, and the transformant of large group has been carried out molecular biology and genetic analysis, studied T-DNA flanking sequence in the paddy rice.After this, utilize the report of agrobacterium-mediated transformation rice transformation success to be on the increase, become the routine techniques of japonica rice genetic transformation.
Maize genetic Study on Transformation progress depends on the improvement of acceptor systems to a great extent.In the later stage in the 1980's, the corn protoplast regenerated plant is successful, is that the genetic transformation work of acceptor launches rapidly with the protoplastis.1988, and Rhodes etc. (Rhodes etc., 1988, Science 240:204-207) obtains transfer-gen plant with electric shocking method, but this plant is sterile.Nineteen ninety, (Gordon-Kamm, WJ. etc., The PlantCell, 2:603-618) transgenic corns that can educate with the acquisition of particle gun blast technique such as Gordon-Kamm.After this, the particle gun blast technique is used more in corn gene.This method acceptor is extensive, can be explant (young fringe, rataria or mature embryo etc.), callus or suspended culture cell etc., operates simplyr, and effect is also better, is widely adopted.
Though the agriculture bacillus mediated genetic transformation of corn begins early, technology waits to improve so far.1987, and Grimsley etc. (Grimsley etc., 1987, Nature, 325:177-179) (maizestreak virus, cDNA MSV) changes corn over to by Agrobacterium to report, and the plant generation systems is infected with corn streak virus.But this research does not show that MSV cDNA energy stable integration is in the corn gene group.1991, and Gould etc. (Gould etc., 1991, Plant Physiol 95:426-434) infects the stripped bud point of corn with Agrobacterium, obtains transfer-gen plant.But the sharp skill that needs of bud of peeling off 0.1mm * 0.3mm is very high, and workload is also very big, and some later investigators fail repetition successfully.
1993, Hiei etc. reported the method for the transforming monocots that a kind of usefulness is agriculture bacillus mediated.This method just adopts (paddy rice and corn) explant in dedifferentiation or dedifferentiation as acceptor, changes HPT and gus gene over to corn (A188) or paddy rice (japonica rice).1996, and Ishida etc. (Ishida etc., 1996, Nature Biotechnol. has reported with Agrobacterium infected maize immature embryos 14:745-750) that the transformation efficiency of A188 self-mating system is 12-30%.But the transformation efficiency of the seed of single cross that derives from A188 is more much lower than A188 (5.3%).Yet Ishida does not obtain transformant in the strain beyond the A188.1999, (Zhao, ZY etc., 1999, US Patent 5981840) such as the Zhao of pioneer species Subsidiary Company obtained the patent of the key self-mating system rataria of Agrobacterium-mediated Transformation corn method.Methods such as this method for transformation and Ishida are close, but the seed of single cross that produces with A188 and other hybridization between selfed lines is a material, and transformation efficiency is brought up to 6.9-50.5% from the 0.4-5.3% of Ishida etc.; With the key self-mating system PHJ90 of corn, PHN46HE and PHP38 is material, and transformation efficiency is 0.9-9.0%, and does not obtain the stable conversion body of these 3 self-mating systems with the method for Ishida etc.
In the same year, China Huang Lu etc. has reported work (Huang Lu etc., 1999, " Journal of Experimental Biology ", 32 (4): 381-389) of adopting embryo callus success of the Agrobacterium-mediated Transformation corn seed of single cross and regeneration plant.Although the existing a large amount of reports of corn gene research, transgenic insect-resistant corn big area is promoted, and is that the work of a large amount of transfer-gen plants of material regeneration is still the bottleneck in the breeding of corn gene engineering with selfing.From the angle of acquisition transfer-gen plant in enormous quantities, still need and set up cost-effective technological method and system.To produce used key selfing is material, and corn tissue and cell cultures difficulty are big, be difficult for bearing a large amount of plant again, and the regeneration plant self-fertility is more difficult.Therefore, overcome genotype barrier, develop that corn culture system and transgenic method are particularly important efficiently.This is a corn gene engineering breeding key link efficiently.
At the beginning of nineteen ninety generation, and Sautter etc. (Sautter, C. etc., 1991, Bio/Technology, 9:1080-1085) and (Vasil, V. etc., 1991, Bio/Technology, 9:743-747 such as Vasil; 1992, Bio/Technology 10:667-674) adopts the particle gun blast technique to obtain the wheat transgenic plant respectively, and Guo Guangqing etc. (Guo Guangqing etc., 1993, " Science Bulletin " 38:1226-1231) adopts the PEG revulsion to obtain the wheat transgenic plant.These work depend on the genotype of material, and the frequency of transformant regeneration plant is all not high.After this, (Altpeter such as Altpeter, F.V. etc., 1996, Nature Biotechnology, 14:1155-1159) and (Barro such as Barro, F. etc., 1997, Nature Biotechnology, 15:1295-1299) respectively high molecular wheat paddy gastral cavity subunit gene is changed in the wheat, transfer-gen plant shows new features.1997, Cheng etc. (Cheng etc., 1997, Plant Physiol., 115:971-980) reported first adopt the work of Agrobacterium success transformed wheat, this work has obtained molecular biology evidence, and the offspring has been carried out genetic analysis.Above-mentioned these work are that material carries out with spring habit or semi-winterness wheat all.The genetic transformation of other wheat crops progress also unhappy (Pawlowski etc., 1998, Proc.Natl.Acad.Sci.U.S.A., 95:12106-12110).By the end of so far, the wheat crops genetic transformation is still a generally acknowledged difficult problem.
In above report, except that minority work, the acquisition of transfer-gen plant all depends on organ, tissue and cell culture technology; The overwhelming majority has the genotype of important economic worth, and can its tissue culture and genetic transformation successfully depend on genotype, particularly cereal crop and big grain leguminous crop to a great extent; Have only minority genotype material to bear large quantities of plant again by tissue and cell cultures.In addition, transformation efficiency is also very low.Especially be acceptor with kind and selfing with commercial value.Can find out also that from above report utilize stripped stem apex or plumule to carry out genetic transformation, not only operation easier is big, efficient is low, and the difficult grasp of conversion condition, and experimental repeatability is poor.Therefore, development is not subjected to the simple and effective genetic transforming method and the system of genotype restriction be significant (Hansen etc., 1999, TrendsPlantSci, 4 (6): 226-231).
Term of the present invention and substratum:
In the present invention, " macroseed plant " mainly is meant cereal crops such as (1) corn, wheat, paddy rice, Chinese sorghum, barley, oat, rye; (2) leguminous crops such as soybean, peanut, mung bean, Kidney bean, string bean; (3) cash crop such as cotton, Sunflower Receptacle." stem apex " is meant ten to tens microns shoot apical meristem (shoot meristemetic) and greatly to tens millimeters bud-end (shoottip), " shoot apical meristem " is meant apical growth awl and contiguous leaf primordium thereof.
In the present invention, the Transformation Program of Agrobacterium (Agrobacterium comprises A.tumefaciens and A.rhizogenes) mediation is basically with (Ishida etc., 1996, Nature Biotechnol., 14:745-750) report such as Ishida.(acetosyringone As) waits phenolic compound and neutral monose to add Syringylethanone in transfection liquid.
In the present invention, the used Agrobacterium of transfection contains improved Ti-plasmid, latter's portability or do not carry herbicide resistance gene or other selective agent resistant gene.
In the present invention, the plant resistance is expressed and given to used herbicide resistance gene or other resistant gene as long as enliven in transformed plant cells, the kind of the source of restriction gene, size, used promotor and institute's antiweed (or other selective agent) is not subjected to the restriction of agrobacterium strains yet.
In the present invention, minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic isoreactivity composition filtration sterilization adds behind medium sterilization.
In the present invention, selective agent such as weedicide sprays with neutral tap water dissolving back.
In the present invention, the general employing of Agrobacterium cultivation YEP substratum (yeast extract 10g/l, peptone 10g/l, NaCl 5g/l, pH 7.0, solid medium adds 15g/l agar) cultivate.
The minimum medium that cereal crop seed germination and embryo culture are adopted is modified MS medium: KNO
31900mg/l, NH
4NO
31650mg/l, CaCl
22H
2O 440mg/l, MgSO
47H
2O 370mg/l, KH
2PO
4H
2O 170mg/l, FeSO
47H
2O 27.8mg/l, ZnSO
47H
2O 10mg/l, MnSO
4H
2O22mg/l, H
3BO
310mg/l, Na
2MoO
40.5mg/l, CuSO
45H
2O 0.05mg/l, CoCl
22H
2O0.05mg/l, vitamin 10mg/l, pyridoxine hydrochloride 1mg/l, nicotinic acid 1mg/l, glycine 2mg/l, inositol 100mg/l, vitamin H 0.05mg/l, casein hydrolysate 250mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8-6.0.The kind of plant growth regulating substance and concentration are adjusted because of the genotype of cultivated material.
The minimum medium that leguminous crop seed germination and embryo culture are adopted is modified MS medium (the same) or improvement B5 medium.Medium component is planted in the back: KNO
32500mg/l, (NH
4)
2SO
4134mg/l, CaCl
22H
2O 150mg/l, MgSO
47H
2O 250mg/l, NaH
2PO
4H
2O 150mg/l, FeSO
47H
2O27.8mg/l, ZnSO
47H
2O 2mg/l, MnSO
4H
2O 10mg/l, H
3BO
310mg/l, Na
2MoO
40.5mg/l, CuSO
45H
2O 0.05mg/l, CoCl
22H
2O 0.05mg/l, vitamin 10mg/l, pyridoxine hydrochloride 1mg/l, nicotinic acid 1mg/l, glycine 2mg/l, inositol 100mg/l, vitamin H 0.05mg/l, casein hydrolysate 250mg/l, sucrose 20g/l, agar powder 7g/l, pH5.8-6.0.The kind of plant growth regulating substance and concentration are adjusted because of the genotype of cultivated material.
In the present invention, transplanted seedling waters 1/2 modified MS medium inorganic salt solution or 1/2 improvement B5 medium inorganic salt solution, pH6.0-7.0 every other day.
Technical scheme of the present invention:
1. set up acceptor systems
Can select following approach respectively according to floristic difference.
(1) obtains the transgene receptor plant by sprouting mature seed.
Get full clean seed (utilizing heterotic crop to select the self-mating system seed of bagging acquisition for use, have the seed of hard sclerotesta or outer fine hair to remove kind of skin or outer fine hair), with 70% alcohol immersion 1-15 minute (because of kind of the subcategory and the situation of carrying disease germs are determined), soaked 10-15 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40 ml water/250 milliliter triangular flask) in the bottle, seals the back and sprouts down at dark condition (15-35 ℃), and note rocking seed.Sprouting temperature and time decides because of kind of a subcategory.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the substratum and continue down to sprout in dark condition.When treating that plumule or plumular axis are stretched to 3-5 centimetre, radicle 2-7 centimetre, peel off coleoptile or cotyledon and 2-3 sheet spire, expose shoot apical meristem and be used for transforming.Should avoid during operation root system is caused damage.
(2) sprout generation transgene receptor plant by mature embryo.
During seed-coat sterilization (having the seed of hard sclerotesta or outer fine hair to remove kind of skin or outer fine hair), earlier with 70% alcohol immersion 1-15 minute (because of kind of the subcategory and the situation of carrying disease germs are determined), soaked 10-15 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-60 ml water/250 milliliter triangular flask) in the bottle, seals the back and places 12-24 hour (the definite time decides because of kind of subcategory and temperature) down at dark condition (22-30 ℃).After treating the abundant imbibition of seed, get embryo with tweezers and be seeded on the substratum, under dark condition, sprout, sprout temperature and be generally 22-30 ℃.The concrete composition of substratum is variant because of floristics.When treating that plumule or plumular axis are elongated to 3-5 centimetre, radicle 2-4 centimetre, peel off coleoptile or cotyledon and 2-3 sheet spire, expose shoot apical meristem and be used for transforming.Should avoid during operation root system is caused damage.
(3) cultivate immature embryo and obtain the transgene receptor plant.
Immature fruit or fruit ear about 1 minute, soak 6-15 minute (because of the material category and the situation of carrying disease germs are determined) with 0.1% mercury chloride, then with sterilized water washing 3-5 time with 70% alcohol immersion again.Constantly rock during the sterilization, thorough to guarantee surface sterilization.Get then that the immature embryo in period carries out isolated culture after the globular embryo.Immature embryo is seeded in the modified MS medium of additional 6-BA (6-benzyl purine) 0.05-0.8mg/l, sucrose concentration 3-5% or improves on the B5 medium and cultivate, and temperature 22-30 ℃, scattered light or dark condition are cultivated down.Medium component changes because of floristics.When treating that plumule or plumular axis are stretched to 3-5 centimetre, radicle 2-4 centimetre, peel off coleoptile or cotyledon and 2-3 sheet spire, the seedling of exposed stem apex is used for transforming.Should avoid during operation root system is caused damage.
(4) with the planting seed of dicotyledons in nursery or flowerpot.
The seedling back lucifuge growth of being unearthed, treat that cotyledon launches after, peel off cotyledon, expose vegetative point, import Agrobacterium with capillary burette or syringe to vegetative point.
2. transform test-tube plantlet or nursery seedling
Getting the seed seedling of exposed shoot apical meristem or the seedling of embryonic development is transformation receptor, goal gene is changed over to the meristematic cell of acceptor plant stem apex with agrobacterium-mediated transformation.Concrete steps are:
(1) preparation transforms and uses carrier.
To have binary vector (Mini-Ti plasmid, have goal gene and/or herbicide resistance gene and/or other selectable marker gene) or Agrobacterium (as EHA101, AGL1 and LBA4404) 28 ℃ of concussion cultivations in additional antibiotic YEP substratum of integrative vector (T-DNA contains in the district goal gene and/or herbicide resistance gene and/or other selectable marker gene) altogether, concussion speed is 110r/min, makes bacterium be in logarithmic phase; Under 3000r/min centrifugal 10 minutes then, abandon supernatant liquor, thalline is with 1/2 improvement MS liquid nutrient medium (being that modified MS medium basal component reduces by half) washing, centrifugal again collection; (acetosyringone, 1/2 improvement MS liquid nutrient medium As) suspends, and dilutes 5-20 and doubly is used for transforming with adding Syringylethanone with thalline again.
(2) adopt Agrobacterium intermediary method to change goal gene over to.
Can adopt following arbitrary program to transform:
A, the shoot apical meristem that aseptic seedling is exposed are put into agrobacterium liquid and are soaked 0.5-8 minute (Agrobacterium is avoided contacting in other position as far as possible), inhale the unnecessary bacterium liquid that removes stem apex with sterilization filter paper, again the plant root is inserted in the solid medium (generally using former substratum), be placed under the dark condition and cultivated (about 22 ℃) 2-4 days, cultivated (22-25 ℃) 2-3 days then under illumination, the existing leaflet in plant top this moment occurs.Again with plantlet of transplant in the flowerpot that is covered with upper strata vermiculite and lower floor's loam.
B, the shoot apical meristem that aseptic seedling is exposed are put into agrobacterium liquid and are soaked 0.5-8 minute (Agrobacterium is avoided contacting in other position as far as possible), inhale the unnecessary bacterium liquid that removes stem apex with sterilization filter paper, then it is transplanted in the flowerpot that is covered with upper strata vermiculite and lower floor's loam, the plant top covers the vermiculite of 2 centimetres of thickness, make it be in the lucifuge state, and keep humidity.Under optimal temperature (18-23 ℃), plant grows young leaves after one week.
C, the seedling in flowerpot or the nursery is peelled off cotyledon, expose vegetative point, with the syringe that has capillary glass-tube or No. 4 (People's Republic of China's entry needle standard) entry needles agrobacterium liquid is injected vegetative point then.The injection back is inhaled with thieving paper and is removed unnecessary drop, covers vermiculite or other soil-less medium of 2 centimetres of thickness then at the plant top, makes it be in the lucifuge state, and keeps humidity.Under optimal temperature (18-23 ℃), plant grows young leaves after a week.
(3) screening of transformed plant, transplanting and management.
For the conversion seedling in flowerpot or the nursery, water 1/2 improvement MS or 1/2 improvement B5 medium inorganic salt every other day, grow 3 young leaves after, spray the weedicide of suitable concentration (killing or suppress the concentration of non-transgenic plant strain growth) or the selective agent of other type.Unconverted plant lacks the specific resistance proterties, and is dead gradually after handling with the selective agent of weedicide or other type.Transfer-gen plant still can be survived and grow behind the selective agent of spraying herbicide or other type because of to weedicide or to the selective agent of other type resistance being arranged.If with herbicide resistance gene als (acetolactate synthase gene) is selective marker, the milpa after the conversion is behind spraying herbicide, and the ratio of resistance individuality can reach more than 30%.Resistant plant length is to 5 leaf phases, with greenhouse or the field of its field planting to fertile soil.Under the suitable growth condition (because of floristics, kind or environmental the variation), transfer-gen plant normal development and solid.For the autophilous plant of non-strictness, note in good time bagging and selfing.
(4) evaluation of transfer-gen plant and filial generation resistance are analyzed.
The blade of getting transformed plant extracts DNA, adopts round pcr to carry out goal gene and detects.Adopt Southern blotting method to identify to the PCR positive plant that obtains to determine transfer-gen plant.Molecular biology identification result shows, is selective marker if use herbicide resistance gene als (acetolactate synthase gene), and the antiweed plant more than 80% is the transgenosis individuality, sees accompanying drawing 1,2.If select the plant of note screening through conversion processing without weedicide or other, directly adopt technology for detection, in the conversion processing individuality, the transfer-gen plant ratio reaches 20-40%.
It is generally acknowledged that genetic transformation can only make in rataria or the meristematic tissue a few cell obtain transforming, if do not utilize selective marker to screen, cell fission forms mosaic, has only the transformant in mosaic to form gamete, and transformation traits just can pass to the offspring.There are how many individualities to carry foreign gene for understanding in the transfer-gen plant filial generation, can be long to 3-5 leaf spraying herbicide or with the selective agent processing plant leaf of other type during the phase at progeny plant.The result shows, adopts the self-bred progeny of the transgenic corns of this technical system acquisition, and almost all there is the transgenosis individuality in each strain system, and in the strain system about 60%, the segregation ratio of foreign gene meets mendelian inheritance.In the present invention, though there is not the step of screening transformant in the Transformation Program, because Agrobacterium for a long time with the meristematic tissue contact, may make than many cells to obtain transforming; When selecting to transform seedling with weedicide, the hyperplasia of non-transformed cell is suppressed, and its ratio reduces rapidly in apical meristem.So that the cell that causes producing reproductive organ nearly all is a transformant.
Beneficial effect of the present invention:
The present invention is a parent material with big grain plant maturation or immature seed, and the sterilization back is sprouted under dark condition or cultivated, and peels off bud scale or cotyledon and spire then, is that intermediary transforms with the Agrobacterium, and then obtains transfer-gen plant.The present invention can effectively reduce genotype barrier, can effectively obtain transfer-gen plant from most genotype; And transfer-gen plant grows normally, and fecundity is good, and its offspring also can keep the feature of protogene type substantially.
Be that the technological method that acceptor carries out genetic transformation is compared with embryo callus and rataria, the advantage that the present invention has is: can allow most genotype produce transfer-gen plants; Experimental period is short; The success ratio height; Remove the use of antibiotics resistance gene from; Draw materials and be not subject to seasonal restrictions.For utilizing heterotic crop, as if being acceptor with selfing, the transfer-gen plant overwhelming majority need not to carry out how just can to produce the transgenosis self-mating system for backcrossing.
Compare for the technological method that acceptor carries out genetic transformation with isolated culture bud point, the advantage that the present invention has is: remove the process of stem tip culture regeneration plant from, simple to operate, transformation efficiency and transformation frequency increase substantially; Good reproducibility; Remove the use of antibiotics resistance gene from, help edible product and identify by security; Transgenosis plant development in the present age is normal, the filial generation of the large quantities of acquisition transfer-gen plants of energy etc.
Embodiments of the invention:
Below narrate embodiments of the invention.What must illustrate is that embodiments of the invention only play illustration to the present invention and effect without limits.
Example one, be intermediary's maize transformation self-mating system aseptic seedling with the Agrobacterium
1. the acquisition of corn aseptic seedling.
Bagging obtains key self-mating system of corn or cross-fertilize seed seed, with 70% alcohol immersion 10 minutes, with 0.1% mercury chloride immersion 10-12 minute, washs 3-5 time with sterilized water then again.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium, under dark condition, continue to sprout.When treating that plumule is stretched to 3-5 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.
2. Agrobacterium is cultivated and activation.
(the Mini-Ti plasmid has weedicide chlorsulfuron resistant gene als will to have binary vector, this gene is from the modification of going forward side by side of the Arabidopis thaliana plant of anti-chlorsulfuron, Li Guosheng etc., 2000, " Science Bulletin ", agrobacterium tumefaciens 45:2181--2184) (AGL1 or LBA4404) 28 ℃ of concussions in additional antibiotic YEP substratum are cultivated, and concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal collection with 1/2 improvement MS liquid nutrient medium.Thalline is suspended with the 1/2 MS improvement liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-20 doubly is used for transforming again.
3. the corn aseptic seedling transforms.
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish, the aseptic seedling top that will expose the stem apex growing tip is immersed in 2-5 minute (AGL12 minute, LBA44045 minute) in the bacterium liquid.
(2) the bud point after the dip-dye blots with aseptic filter paper, root is inserted in the modified MS medium cultivated 2-3 days in dark, and culture temperature is 22-24 ℃, then aseptic seedling is placed under the scattered light to cultivate 2 days.
(3) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot that is covered with upper strata vermiculite and lower floor's loam, vermiculite covers the plant top.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
4. transformed plant screening and field planting
After transformed plant grows 3 leaves, spraying 20mg/l chlorsulfuron (Shenyang Agricultural Chemicals Factory produces, the effective constituent 25%) aqueous solution, is contrast with unconverted plant.The sprinkling amount is fallen drop with plant and is advisable.Adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 10 days.Plant after the conversion processing, the variation of some individualities is similar to adjoining tree, and other individualities then continue growth, no considerable change.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.
5. the evaluation of transfer-gen plant
The antiweed plant strain growth is got blade and is extracted DNA during to the 7-8 leaf, adopts round pcr to detect foreign gene, the PCR positive plant is carried out Southern blotting detect.The results are shown in accompanying drawing 1,2,3.The result shows that about 86% antiweed plant is the transgenosis individuality.
6. the management of transfer-gen plant and progeny analysis
Bagging selfing or sisters handed over solid after transfer-gen plant was bloomed.Planting seed is in the land for growing field crops or the greenhouse, and plant is long gets blade to the 4-6 leaf and extract DNA during the phase, adopt round pcr to detect and whether have foreign gene, and add up the segregation ratio of foreign gene in progeny plant.The results are shown in Table 1.
Table 1, the frequency of foreign gene als in the transfer-gen plant filial generation
| Strain system | The progeny plant number | PCR positive plant number | Male/female strain ratio | Approximate segregation ratio male/female | Whether meet mendel's law |
| TA-01 | 35 | 27 | 27/8 | 3/1 | Be |
| TA-02 | 30 | 28 | 28/2 | 14/1 | Be |
| TA-04 | 33 | 25 | 25/8 | 3/1 | Be |
| TA-06 | 32 | 32 | 32/0 | 1/0 | Undetermined |
| TA-08 | 30 | 28 | 28/2 | 14/1 | Be |
| TA-10 | 29 | 20 | 20/9 | 2/1 | Not |
| TA-12 | 30 | 27 | 27/3 | 9/1 | Not |
| TA-14 | 31 | 30 | 30/1 | 30/1 | Undetermined |
| TA-16 | 32 | 30 | 30/2 | 15/1 | Be |
| TA-18 | 30 | 21 | 21/9 | 2/1 | Not |
| TA-20 | 43 | 40 | 40/3 | 13/1 | Be |
| TA-22 | 32 | 25 | 25/7 | 3/1 | Be |
| TA-24 | 30 | 28 | 28/2 | 14/1 | Be |
| TA-26 | 30 | 19 | 19/11 | 2/1 | Not |
| TA-28 | 50 | 47 | 47/3 | 15/1 | Be |
| TA-30 | 31 | 7 | 7/24 | 1/3 | Not |
| TA-32 | 30 | 30 | 30/0 | 1/0 | Undetermined |
| TA-34 | 31 | 26 | 26/5 | 5/1 | Not |
| TA-36 | 34 | 32 | 32/2 | 16/1 | Be |
| TA--38 | 29 | 27 | 27/2 | 14/1 | Be |
Data show, are genetically modified receptor tissue with aseptic seedling stem apex meristematic tissue, can efficiently obtain transfer-gen plant, and the segregation ratio majority of foreign gene in progeny plant meets mendel's law.
Example two, be that intermediary transforms the aseptic seedling that is produced by the corn immature embryo with the Agrobacterium
1. the corn aseptic seedling obtains
Get the key self-mating system selfing of 15-20 days the corn in bagging pollination back fruit ear, remove behind the bract with 70% alcohol immersion 5-8 minute, then with the scalpel seed top of pruning, choose immature embryo with tweezers, be seeded in additional 0.25mg/l 2, on the modified MS medium of 4-D (2,4 dichlorophenoxyacetic acid), cultivate down at dark condition (23-26 ℃).When treating that plumule is stretched to 3-5 centimetre, peel off coleoptile and 2-3 sheet spire, expose the stem apex growing tip.Adopt Agrobacterium intermediary method to transform shoot apical meristem then.
2. all the other operation stepss comprise that Agrobacterium cultivation and activation, the conversion of corn aseptic seedling, transformed plant screening, field planting and molecular biology identification etc. are all identical with embodiment one.
Example three, be intermediary's transformed wheat aseptic seedling with the Agrobacterium
1. the wheat aseptic seedling obtains
The seed of wheat improved seeds with 70% alcohol immersion 1-3 minute, soaked 8-12 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40 milliliter/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium and sprout down in dark condition.When treating that the plumule elongation ends 3-5 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.
2. Agrobacterium is cultivated and activation
To have the binary vector agrobacterium tumefaciens of (the Mini-Ti plasmid has herbicide resistance gene bar) (AGL1 or LBA4404) 28 ℃ of concussion cultivations down in additional antibiotic LB substratum, concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs with 1/2 MS liquid nutrient medium, centrifugal collection.Thalline is suspended with the 1/2 MS liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-20 doubly is used for transforming again.
3. the wheat aseptic seedling transforms
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish makes the aseptic seedling top of exposing the stem apex growing tip be immersed in the bacterium liquid 3-6 minute.
(2) the bud point after the dip-dye blots with aseptic filter paper, root is inserted on the modified MS medium of adding the 200mg/l glutamine cultivated 2-3 days in dark, and culture temperature is 22-24 ℃.Then aseptic seedling is placed under the scattered light and cultivated 2 days.
(3) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, and covers the plant top with vermiculite.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 15-21 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
4. transformed plant screening and field planting
After transformed plant grows 3 leaves, and spraying herbicide phosphinothricin (Sigma company, St.Louis, MO, USA) the 2mg/l aqueous solution falls drop with plant and is advisable.Unconverted adjoining tree stops growing after back 5 days in sprinkling, and beginning is dead about 15 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individual continuing grow, and change not obvious.When the plant of waiting to survive length arrives the 7-8 leaf, the field is arrived in its field planting, and promoted to tiller.
5. the antiweed plant is after vernalization treatment, standing up-jointing stage gets blade and extracts DNA, adopts round pcr to detect transformed plant.The PCR positive plant carries out Southern blotting and detects, and in the 368 strain antiweed plant that obtain, about about 80% (296/368) are the transgenosis individuality.
6. transfer-gen plant progeny analysis
Plant by vernalization stage (the subzero treatment condition is different because of kind) was stood up under the long day, jointing, blossom and bear fruit.The seed that transfer-gen plant produces is put after immersion is sprouted in the refrigerator (0-2 ℃) and was carried out vernalization treatment 30-65 days.Seed after part vernalization treatment phosphinothricin (Sigma company, St.Louis, MO, USA) the 2mg/l aqueous solution is handled, and observes the individual ratio of statistics resistance and susceptibility; Planting seed after another part vernalization treatment is in the land for growing field crops or the greenhouse, treats that plant is long to get blade during the phase to the 5-6 leaf and extract DNA, adopts round pcr to detect foreign gene, and adds up the segregation ratio of foreign gene in progeny plant.Resistance screening and molecular Biological Detection result show that all in the strain system of about half, the segregation ratio of foreign gene in progeny plant meets mendel's law.
Example four, be intermediary's converting cotton aseptic seedling with the Agrobacterium
1. the cotton aseptic seedling obtains
Get the seed of cotton self-mating system or improved seeds, slough surperficial fine hair,, soaked 10-12 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time with 70% alcohol immersion 2 minutes with the vitriol oil.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium that ammonium salt reduces by half and sprout down in dark condition.When treating that plumular axis is stretched to 3-5 centimetre, peel off cotyledon and spire, emergent stem end growing tip.
2. Agrobacterium is cultivated and activation
To have agrobacterium tumefaciens (AGL1 or LBA4404) 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum of binary vector (the Mini-Ti plasmid has weedicide chlorsulfuron resistant gene als), concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under the 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.The 1/2 improvement MS liquid nutrient medium washing that thalline reduces by half with ammonium salt, centrifugal collection.Thalline is suspended with the 1/2 MS liquid nutrient medium that the ammonium salt that adds the 100mg/l Syringylethanone reduces by half, dilution 10-25 doubly is used for transforming again.
3. the cotton aseptic seedling transforms
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish makes the shoot apex that exposes the stem apex growing tip be immersed in the bacterium liquid 2-5 minute.
(2) the bud point after the dip-dye blots with aseptic filter paper, and cultivating 2-3 days in dark on the modified MS medium of the no growth regulator of root insertion, culture temperature is 24-26 ℃.Then aseptic seedling is placed under the scattered light and cultivated 2 days.
(3) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot of upper strata vermiculite lower floor loam, vermiculite covers the plant top.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
4. transformed plant screening and field planting
After transformed plant grows 3 leaves, spray 1.5mg/l chlorsulfuron (Shenyang Agricultural Chemicals Factory produces, the effective constituent 25%) aqueous solution, fall drop with plant and be advisable.Unconverted adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 12 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individualities have very strong resistance, continue growth.The survival plant is long during the phase, to arrive field with its field planting to 5 leaves.
5. the molecular biology identification of antiweed plant
When antiweed plant length arrives the 7-8 leaf, get blade and extract DNA, adopt round pcr to detect foreign gene.The PCR positive plant carries out Southern blotting and detects.In the 186 strain antiweed PCR positive plants that obtain, about more than 80% the plant of (157/186) be the transgenosis individuality.
6. the molecular biology identification of transfer-gen plant filial generation
Selfing or sisters handed over solid after transfer-gen plant was bloomed.Planting seed is got blade at plant 4-6 leaf and is extracted DNA in the land for growing field crops or the greenhouse during phase, adopt round pcr to detect progeny plant and whether have foreign gene, and the statistics foreign gene is at the filial generation segregation ratio.The result shows, is the transgene receptor tissue with aseptic seedling stem apex meristematic tissue, can efficiently obtain transfer-gen plant.And in the transgenic line of (23/38), the segregation ratio majority of foreign gene in progeny plant meets Mendelism about 60%.
Example five, be intermediary's soybean transformation aseptic seedling with the Agrobacterium
1. the soybean aseptic seedling obtains
Get the seed of improved seeds,, soaked 10-12 minute, then with sterilized water washing 3-5 time with 0.1% mercury chloride with 70% alcohol immersion 2-3 minute.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (50-60 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 2-3 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the improvement B5 medium and sprout down in dark condition.When treating that plumular axis grows to 3-5 centimetre, peel off cotyledon and emergent stem end growing tip.
2. Agrobacterium is cultivated and activation
To have agrobacterium tumefaciens (AGL1 or LBA4404) 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum of binary vector (the Mini-Ti plasmid has weedicide chlorsulfuron resistant gene als), concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under the 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal again collection with 1/2 improvement B5 liquid nutrient medium (promptly improveing B5 medium basal component reduces by half).Thalline is suspended with the 1/2 improvement B5 liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-10 doubly is used for transforming again.
3. the soybean aseptic seedling transforms
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish makes the aseptic seedling top of exposing the stem apex growing tip be immersed in the bacterium liquid 3-6 minute.
(2) the bud point after the dip-dye blots with aseptic filter paper, the seedling butt is inserted on the improvement B5 medium cultivated 3-4 days in dark, and culture temperature is 22-24 ℃.
(3) aseptic seedling is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, the top covers vermiculite, and natural lighting is growth down, warm 23-28 ℃ of day, at temperature 20-25 ℃ of night, waters 1/2 improvement B5 medium inorganic salt every other day.
4. transformed plant screening and field planting
After transformed plant grows 3 leaves, spray 2.0mg/l (because of the genotype difference) chlorsulfuron (Shenyang Agricultural Chemicals Factory produces, the effective constituent 25%) aqueous solution, fall drop with plant and be advisable, unconverted adjoining tree stopped growing in sprinkling in back 4 days, and beginning is dead about 15 days.Transformed plant is after sprinkling, and the variation of some individualities is similar to adjoining tree, and other individual continuing grow, and change not obvious.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.
5. when the antiweed plant strain growth is to the 7-8 leaf, gets blade and extract DNA, adopt round pcr to detect foreign gene.The PCR positive plant carries out Southern blotting and detects.In the 253 strain antiweed PCR positive plants that obtain, about more than 60% the plant of (156/253) be the transgenosis individuality.
6. the molecular biology identification of transfer-gen plant filial generation
The seed that transfer-gen plant produces is sowed in the land for growing field crops or the greenhouse.Plant 3-4 leaf is got blade and is extracted DNA during the phase, adopt round pcr to detect foreign gene, and the segregation ratio of statistics foreign gene in progeny plant.The result shows, is the transgene receptor tissue with aseptic seedling stem apex meristematic tissue, can efficiently obtain transfer-gen plant.In the strain system of about half (16/30), the segregation ratio of foreign gene in progeny plant meets mendel's law.
The Figure of description explanation:
The PCR result of Fig. 1, antiweed transgenosis (als) plant:
Wherein, 1,11 is GeneRuler
TMDNALadderMix, the positive result of 2-8,9 for plasmid PCR ties (showing the 1.1kb band) really, 10 negative contrasts.
Fig. 2: the Southern results of hybridization of antiweed transgenosis (als) plant
Wherein, the positive result of 1-5,6 negative contrasts, 7 are plasmid DNA/EcoRI (showing the 2.5kb band), 8 is λ DNA/HindIII.
Fig. 3: antiweed transgenosis (als) regeneration plant
To transgenosis seedling spraying concentration is the chlorsulfuron solution of 40mg/L, and the growth of seedling is unaffected, and transfer-gen plant is strong to the tolerance of weedicide chlorsulfuron.
Claims (14)
1. the method for a transforming macroseed plant is characterized in that this method mainly may further comprise the steps:
(1) gets large seed or mature embryo or immature embryo and sprout, obtain the acceptor plant;
(2) the acceptor plant with childhood is a material, peels off bud scale or cotyledon and spire on suitable opportunity, exposes the direct acceptor of stem apex as genetic transformation;
(3) with the stem apex of Agrobacterium intermediary method transformation receptor plant, goal gene is imported the shoot apical meristem cell;
(4) spray selective agent after the plant after the conversion grows 3-4 sheet young leaves, filter out the transfer-gen plant of performance resistance;
(5) resistance and molecular Evidence detection are carried out in the filial generation of transfer-gen plant, selected the transgenosis individuality.
2. the process of claim 1 wherein that said macroseed plant is the cereal crop plant.
3. the process of claim 1 wherein that said macroseed plant is corn, wheat, paddy rice, Chinese sorghum, barley, oat, rye.
4. the process of claim 1 wherein that said macroseed plant is the leguminous crop plant.
5. the process of claim 1 wherein that said macroseed plant is soybean, peanut, mung bean, Kidney bean, pea, string bean, broad bean, kidney bean.
6. the process of claim 1 wherein that said macroseed plant is economic class crop plants.
7. the process of claim 1 wherein that said macroseed plant is cotton, Sunflower Receptacle, castor-oil plant.
8. the approach of said " obtaining the acceptor plant " that the process of claim 1 wherein can be selected respectively according to floristic difference, has:
(1) obtains the transgene receptor plant by sprouting mature seed;
(2) embryo germination of getting mature seed produces the transgene receptor plant;
(3) cultivating immature embryo makes it sprout acquisition transgene receptor plant;
(4) with the planting seed of dicotyledons in flowerpot or nursery.
9. the process of claim 1 wherein that said " suitable opportunity " is different because of floristics, and to be in when Agrobacterium is best competence with growing tips of the plant be the best.
10. the process of claim 1 wherein that said " Agrobacterium intermediary method " comprises infusion method, dips method, injection, ultrasonication-agroinfection, ray processing-agroinfection, agroinfection-vacuum introductory technique.
11. the process of claim 1 wherein that said " selective agent " is weedicide.
12. a method of producing the transfer-gen plant filial generation of macroseed plant is characterized in that this method application rights requires the method for 1 transforming macroseed plant.
13. a method that produces the transgenosis self-mating system of macroseed plant is characterized in that this method application rights requires the method for 1 transforming macroseed plant.
14. a method of avoiding using the transforming macroseed plant of antibiotics resistance gene is characterized in that this method application rights requires the method for 1 transforming macroseed plant.
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| CN100360677C (en) * | 2005-05-13 | 2008-01-09 | 吉林省农业科学院 | Transgene method for plant |
| WO2010146046A1 (en) * | 2009-06-15 | 2010-12-23 | Basf Plant Science Company Gmbh | Hydroponic systems for generating transgenic plants |
| CN102229947B (en) * | 2011-05-31 | 2012-10-03 | 山西省农业科学院棉花研究所 | Method for directly transforming cotton seed embryos by utilizing agrobacterium tumefaciens |
| CN103667342B (en) * | 2013-11-29 | 2015-10-28 | 河南科技学院 | A kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton |
| CN105420274B (en) * | 2015-12-07 | 2019-06-28 | 中国农业大学 | A kind of corn mature embryo stem apex method for transformation of mediated by agriculture bacillus |
| CN108165572A (en) * | 2017-11-07 | 2018-06-15 | 河南农业大学 | A kind of agriculture bacillus mediated needle thorn growing point transformed wheat method |
| CN108546710A (en) * | 2018-04-20 | 2018-09-18 | 刘寒冬 | A kind of castor-oil plant genetic transforming method |
| CN109652438A (en) * | 2018-12-25 | 2019-04-19 | 河南农业大学 | A kind of Wheat genetic transformation method based on wheat seed embryo injection Agrobacterium |
| CN113604497B (en) * | 2021-07-28 | 2023-03-24 | 浙江大学 | Genetic transformation method of gramineous plants |
| CN116445537A (en) * | 2023-06-19 | 2023-07-18 | 海南大学三亚南繁研究院 | Genetic transformation method of dragon fruit |
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